The objective of this proposal is to contribute to an understanding of the features in eukaryotic messenger RNA which influence ribosome binding, by analyzing and manipulating the initiation regions from six reovirus messenger RNAs. The general strategy is to make use of 5'-terminal reovirus RNA fragments of known nucleotide sequence, which will be further characterized with respect to (1) identification of the viral genome segment that corresponds to each initiation region, (2) identification within each fragment of the AUG codon used to initiate translation, (3) determination of the relative efficiency of ribosome binding to each fragment, and (4) analysis of the secondary and tertiary conformation of the 5'-terminal RNA fragments. In addition, the sequence of certain fragments will be manipulated (using enzymatic and chemical agents) in order to test a number of specific hypotheses regarding features that contribute to selection of these regions of mRNA by ribosomes. The possibility of a four base-pair interaction between the initiator tRNA and reovirus mRNA is one of the hypotheses to be tested. Based on earlier work with reovirus and other eukaryotic messages, a "scanning mechanism" was proposed to explain how eukaryotic ribosomes select initiation regions in messenger RNA. The model postulates that a 40S ribosomal subunit binds initially at or near the 5' end of mRNA and subsequently migrates along the RNA chain until the first AUG codon is encountered, whereupon the 40S subunit stops, a 60S subunit joins and translation begins. This idea will be tested both by introducing specific alterations in the sequences of certain reovirus messages and, more critically, by constructing synthetic ribopolymers of defined sequence.